Definitions; Here we try to briefly explain the definition of the terms used but this is not limited to its vast definition.
Particles: can be porous or non-porous and be of any shape and size.
Metal oxide: Oxides of individual metals or mixed metal oxides, which contain more than one metal. Metal oxides may contain other elements or functional groups.
Modification of metal oxides: means that the surface density or number of active centers, which binds negative charged molecules, are reduced by some chemical reactions or physical means such as coating, covering, non covalent interaction.
Biomolecules: The molecules of biological source can be further modified or fragmented. This is not limited to proteins, peptides, DNA, RNA, lipids, small molecules such as vitamins, carbohydrates, oligosaccharides, and combination of these molecules.
In recent years, the proteomics field is growing very rapidly to elucidate the structure of proteins. The post translation modifications (PTM) of proteins, by attaching the groups such as phosphate, sulfate, acetate, oxalate, carbohydrates, lipids, and many other functional groups, make these proteins to biological functional proteins. The study of these PTM's is very important for understanding and study of the biological functions of the biomolecules. One of the most important PTM is the attachment of phosphate group on the proteins. The position of the phosphate group can be achieved by fragmenting the proteins by using proteases. After fragmentation of proteins, the peptides are formed which can be analyzed by Mass spectrometer, directly or after separation on HPLC (High performance Liquid Chromatography. The enrichment of such modified peptides is very important, because sometime, these peptides concentration is too low and can not be identified if large amount of non specific or unmodified peptides are present. For further analysis of the small amounts of these PTM peptides, enrichment and purification steps are needed. In recent years, there are a no. of publications on the applications of metal oxides such as TiO2 or ZrO2 for the enrichment of the phosphopeptides. However, there is a drawback in using the pure metal oxides since sometimes the multi phosphate groups in the same peptides bind so strongly that they can not be eluted back. It is also possible that other negatively charged functional groups such as carboxyl and/or sulfate or any other negatively charged groups can interact with the metal oxide and create a binding stronger then the phosphate group itself.
To overcome the above problem, here we describe the use of modified metal oxides for selective binding of the phospho group containing molecules. For example, the phospho-peptides or proteins can be easily purified by using the TiO2 or ZrO2. However, the phospho-proteins or -peptides also contain besides the phospho group, other negatively charged groups such as carboxyl, sulfate etc. These negatively charged groups also bind with the metal oxides. The bound negatively charged molecules on the metal oxide can be eluted from the metal oxide particles by using high pH or changing the buffer or solution conditions. If more than one negative charge is present, the binding is stronger and it is difficult to elute the phospho molecule from the metal oxide particles. Here we show the use of the modified metal oxides in which case the modifications reduce the density of the Lewis acid centers of the metal oxide particles. This also allows the selective binding of the phospho group and other negatively charged molecules. The interaction between the negatively charged molecules is reduced due to a lower no. of active centers (the density of the Lewis acid groups) at metal oxide. Therefore, the binding occurs between the Lewis acid group on metal oxide and phospho group of the molecule. Other negatively charged molecules, which do not contain the phospho group may not bind under the same condition in which the phosphate group binds.
Furthermore, once the active center is reduced at the metal oxide, the multi phospho groups of the biomolecules will not bind at multiple Lewis acid centers at the metal oxide. This will enable the elution of the phospho molecule from the metal oxide under less drastic conditions. This will also further enable the purification of more selective phospho molecules from other negatively charged molecules.
The various features of novelty, which characterize the above invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its advantages and objects, reference is made to the accompanying drawings and descriptive matter in which a preferred embodiment of the invention is illustrated.